Project Summary Breast cancer is the most common malignancy and the second leading cause of female cancer mortality in the US. Despite the availability of endocrine therapy, chemotherapeutics, and biologic agents, metastatic breast cancer remains incurable. Oncolytic virotherapy lacks cross resistance with conventional systemic interventions for breast cancer, and accumulating preclinical and clinical evidence suggests that viral cell death induced antitumor immunity may contribute to the therapeutic effects of these viral vectors. The oncolytic potential of wild type measles virus (MV) was first recognized following the publication of a number of case reports documenting regression of various hematologic malignancies after natural MV infection. In order to avoid potential complications associated with use of wild type MV strains, we pioneered the use of engineered strains of Edmonston MV vaccine lineage in cancer treatment. Early clinical testing of MV strains encoding for the sodium iodine symporter (NIS) marker gene (MV-NIS) by our group showed clinical activity in advanced malignancies with development of antitumor immune response. Parallel work testing of MV strains in breast cancer lines and animal models demonstrated significant antitumor activity, associated with tumor cell infection and viral replication, independent of ER/PR and HER2 receptor status, To enhance the immunotherapeutic potential of this oncolytic virotherapy approach, we engineered the MV vaccine strain to express an immunostimulatory transgene, the neutrophil activating protein of H. pylori (NAP), a potent toll-like receptor 2 agonist, and we demonstrated superior antitumor efficacy of NAP encoding MV strains. Effector phase immune responses may be dampened by immune-suppressive mechanisms among which the PD-1/PD-L1 axis plays a prominent role. With growing evidence of the antitumor activity of single agent anti-PD-1 agents in refractory breast cancer patients, there is significant potential for immune checkpoint blockade to augment the promising antitumor activity of oncolytic immunovirotherapy in breast cancer. We therefore hypothesize that combining MV-s-NAP with PD-1 blockade will be synergistic and superior to either agent alone in the treatment of breast cancer. In Specific Aim 1, we will conduct the first in-human phase I clinical trial of single- agent intratumoral measles virus (MV-s-NAP) in metastatic breast cancer. In Specific Aim 2, we will evaluate the efficacy, optimal sequence and mechanism of action of MV virotherapy in conjunction with antibody blockade of the PD-1/PD-L1 axis in immunocompetent breast cancer models; and conduct preclinical toxicology studies to evaluate the safety of combination therapy in measles replication permissive transgenic models and assess biodistribution. In Specific Aim 3, we will conduct the first phase I clinical trial to test the combination of intratumoral MV-s-NAP and PD-1 blockade in patients with metastatic breast cancer. The primary clinical endpoints of the two phase I clinical trials are to (a) evaluate safety and tolerability and (b) identify the maximum tolerated dose (MTD); in addition, tissue and blood samples will be collected to assess for systemic antitumor immune response with MV alone and in combination with checkpoint blockade. The proposed series of preclinical and clinical studies will have significant impact by introducing a novel therapeutic approach to the armamentarium against breast cancer.